PKC Inhibition Improves Human Penile Vascular Function and the NO/cGMP Pathway in Diabetic Erectile Dysfunction: The Role of NADPH Oxidase

Abstract

Erectile dysfunction (ED) is a frequent and difficult-to-treat condition in diabetic men. Protein kinase C (PKC) is involved in diabetes-related vascular and cavernosal alterations. We aimed to evaluate the role of PKC in endothelial dysfunction and NO/cGMP impairment associated with diabetic ED in the human corpus cavernosum (CC) and penile resistance arteries (PRAs) and the potential mechanisms involved. Functional responses were determined in the CC and PRAs in patients with non-diabetic ED and diabetic ED undergoing penile prosthesis insertion. PKC activator 12,13-phorbol-dibutyrate (PDBu) impaired endothelial relaxations and cGMP generation in response to acetylcholine in the CC from non-diabetic ED. PDBu also impaired responses to a PDE5 inhibitor, sildenafil, in non-diabetic ED patients. Conversely, a PKC inhibitor, GF109203X, improved endothelial, neurogenic, and PDE5-inhibitor-induced relaxations and cGMP generation only in the CC in diabetic ED patients. Endothelial and PDE5-inhibitor-induced vasodilations of PRAs were potentiated only in diabetes. Improvements in endothelial function in diabetes were also achieved with a specific inhibitor of the PKCβ2 isoform or an NADPH-oxidase inhibitor, apocynin, which prevented PDBu-induced impairment in non-diabetic patients. PKC inhibition counteracted NO/cGMP impairment and endothelial dysfunction in diabetes-related ED, potentially improving response to PDE5 inhibition.

Keywords: NADPH oxidase; NO/cGMP pathway; diabetes; endothelial dysfunction; erectile dysfunction; human corpus cavernosum; human penile arteries; protein kinase C.

Figure 1

The effects of PKC inhibition on the improvement of endothelial relaxation in the human corpus cavernosum (HCC) were specifically related to the presence of diabetes. The upper panels show the effects of PKC activation with 12,13-phorbol dibutyrate (PDBu, 0.3 µM) on endothelial relaxations in response to acetylcholine (ACh, 1 nM to 10 µM) in the HCC in non-diabetic ED patients (ED NoDM) (A) and in diabetic patients with ED (ED DM) (B). The lower panels show the effects of PKC inhibition with GF109203X (1 µM) on ACh-induced responses in the HCC in ED NoDM (C) and ED DM (D). All tissues were precontracted with phenylephrine (PE, 1–10 µM). Data are expressed as the mean ± S.E.M of the percentage of relaxation. n indicates the number of patients from whom the tissues were collected. *** p < 0.001 according to a two-factor ANOVA. (E) shows the effects of GF109203X (1 µM) on the cGMP accumulation driven by ACh (10 µM) in the HCC in ED NoDM and ED DM. Data are expressed as the mean ± S.E.M. of the pmoles of cGMP normalized by the protein content of the tissue. Numbers of patients are indicated in parentheses. * p < 0.05 vs. ED NoDM, †† p < 0.01 vs. the respective data without GF109203X according to the Kruskal–Wallis test followed by Dunn’s post-hoc test.

Figure 2

Endothelial vasodilation of human penile resistance arteries (HPRAs) in ED patients was improved through PKC inhibition only in diabetic patients. (A) shows the effects of PKC activation with 12,13-phorbol dibutyrate (PDBu, 0.3 µM) on endothelial vasodilations in response to acetylcholine (Ach, 1 nM to 10 µM) in the HPRAs of non-diabetic ED patients (ED NoDM). (B,C) show the effects of PKC inhibition with GF109203X (1 µM) on ACh-induced responses in the HPRAs of ED NoDM and diabetic ED patients (ED DM), respectively. (D) shows the influence of NO synthase and cyclooxygenase inhibition with N^G-nitro-L-arginine methyl ester (L-NAME, 100 µM) and indomethacin (INDO, 10 µM), respectively, on the effects of GF109203X on ACh-induced responses in the HPRAs of ED DM patients. All vascular preparations were precontracted with norepinephrine (NE, 1–10 µM). Data are expressed as the mean ± S.E.M of the percentage of relaxation. n indicates the number of patients from whom the tissues were collected. *** p < 0.001 according to a two-factor ANOVA.

Figure 3

Nitrergic relaxation of the human corpus cavernosum (HCC) was enhanced by PKC inhibition specifically in diabetic patients. Effects of PKC activation with 12,13-phorbol dibutyrate (PDBu, 0.3 µM) on neurogenic nitrergic relaxations induced by electrical field stimulation (EFS, 0.5 to 16 Hz) in the HCC of non-diabetic patients with ED (ED NoDM) (A) and effects of PKC inhibition with GF109203X (1 µM) on nitrergic relaxations in the HCC of ED NoDM (B) and diabetic patients with ED (ED DM) (C). All tissues were precontracted with phenylephrine (PE, 1–10 µM). Data are expressed as the mean ± S.E.M of the percentage of relaxation. n indicates the number of patients from whom the tissues were collected. *** p < 0.001 according to a two-factor ANOVA.

Figure 4

PKC inhibition enhanced the efficacy of PDE5 inhibition in relaxing the human corpus cavernosum and accumulating cGMP in diabetes. The effects of PKC activation with 12,13-phorbol dibutyrate (PDBu, 0.3 µM) (A,B) and of PKC inhibition with GF109203X (1 µM) (C,D) on relaxations induced by the PDE5 inhibitor sildenafil (1 nM to 1 µM) in the HCC of non-diabetic patients with ED (ED NoDM) (A,C) and diabetic patients with ED (ED DM) (B,D). (E) shows the effect of the PKC inhibitor on relaxations induced by the PDE5 inhibitor tadalafil (1 nM to 1 µM) in the HCC of ED DM patients. All tissues were precontracted with phenylephrine (PE, 1–10 µM). Data are expressed as the mean ± S.E.M of the percentage of relaxation. n indicates the number of patients from whom the tissues were collected. *** p < 0.001 according to a two-factor ANOVA. (F) shows the effects of GF109203X (1 µM) on the cGMP accumulation driven by sildenafil (SILD, 1 µM) in the HCC of ED NoDM and ED DM patients. Data are expressed as the mean ± S.E.M. of the pmoles of cGMP normalized by the protein content of the tissue. Numbers of patients are indicated in parentheses. * p < 0.05 vs. ED NoDM, † p < 0.05 vs. the respective data without GF109203X according to the Kruskal–Wallis test followed by Dunn’s post-hoc test.

Figure 5

PKC inhibition enhanced the efficacy of PDE5 inhibition in causing vasodilation of human penile resistance arteries (HPRAs) in diabetes. Effects of PKC inhibition with GF109203X (1 µM) on vasodilations induced by the PDE5 inhibitor sildenafil (1 nM to 10 µM) in the HPRAs of non-diabetic patients with ED (ED NoDM) (A) and diabetic patients with ED (ED DM) (B). (C) shows the effect of the PKC inhibitor on relaxations induced by the PDE5 inhibitor tadalafil (1 nM to 100 µM) in the HCC of ED DM patients. All vascular preparations were precontracted with norepinephrine (NE, 1–10 µM). Data are expressed as the mean ± S.E.M of the percentage of relaxation. n indicates the number of patients from whom the tissues were collected. *** p < 0.001 according to a two-factor ANOVA.

Figure 6

Specific inhibition of the PKCβ2 isoform improved endothelial relaxation of penile vascular tissues in diabetes. Effects of PKCβ2 inhibition with CGP53353 (0.3 µM) on endothelial relaxations in response to acetylcholine (ACh, 1 nM to 10 µM) in the human corpus cavernosum (HCC) (A) and penile resistance arteries (HPRAs) (B) of diabetic patients with ED (ED DM). HCC strips were precontracted with phenylephrine (PE, 1–10 µM), while HPRA segments were precontracted with norepinephrine (NE, 1–10 µM). Data are expressed as the mean ± S.E.M of the percentage of relaxation. n indicates the number of patients from whom the tissues were collected. *** p < 0.001 according to a two-factor ANOVA.

Figure 7

Inhibition of NADPH-oxidase improved endothelial relaxation and cGMP accumulation in the penile vascular tissues of diabetic patients and prevented the impairment of endothelial relaxations driven by PKC activation in non-diabetic ED. Effects of NADPH-oxidase inhibition with apocynin (APOC, 10 µM) on endothelial relaxations in response to acetylcholine (ACh, 1 nM to 10 µM) in the human corpus cavernosum (HCC) (A–C) and penile resistance arteries (HPRAs) (D–F) of non-diabetic patients with ED (ED NoDM) (A,D) and diabetic patients with ED (ED DM) (C,F). Panels B and E show the influence of the NADPH-oxidase inhibitor on the impairment driven by the PKC activator 12,13-phorbol dibutyrate (PDBu 0.3 µM) on endothelial relaxations induced by ACh in the HCC (B) and HPRAs (E) of ED NoDM patients. HCC strips were precontracted with phenylephrine (PE, 1–10 µM), while HPRA segments were precontracted with norepinephrine (NE, 1–10 µM). Data are expressed as the mean ± S.E.M of the percentage of relaxation. n indicates the number of patients from whom the tissues were collected. *** p < 0.001 according to a two-factor ANOVA. (G) shows the effects of APOC (10 µM) on the cGMP accumulation driven by ACh (10 µM) in the HCC of ED DM patients. Data are expressed as the mean ± S.E.M. of the pmoles of cGMP normalized by the protein content of the tissue. n indicates the number of patients. ** p < 0.01 according to the Mann–Whitney U-test.

Figure 8

Schematic representation of the potential effects of PKC inhibition on diabetic erectile dysfunction. Nitric oxide released upon sexual stimulation from the endothelium (through endothelial nitric oxide synthase, eNOS) and nitrergic nerves (through neuronal NOS, nNOS) stimulates soluble guanylyl cyclase (sGC) in penile smooth muscle, which increases cyclic guanosine monophosphate (↑cGMP), leading to relaxation of the smooth muscle and penile erection. Type 5 phosphodiesterase (PDE5) is the main enzyme regulating the hydrolysis of cGMP to GMP in penile smooth muscle. PDE5 inhibitors (−) such as sildenafil amplify NO-mediated signaling by slowing cGMP degradation and represent the first-line therapy for erectile dysfunction (ED). However, these drugs require some level of NO availability for their therapeutic effect, which might be compromised in diabetic ED, increasing the risk of treatment failure. The impact of diabetes seems to be related to increased protein kinase C-β (↑PKC-β) activity, since the inhibition of PKC with GF109203X, and, specifically, PKC-β with CGP53353 results in enhanced NO-mediated functional responses, including PDE5-inhibitor-induced relaxation, only in penile tissues from diabetic patients. PKC would increase the generation of reactive oxygen species (ROSs) by NADPH-oxidase (NOX), reducing NO bioavailability and, thus, compromising penile erection. This is supported by the ability of the NOX inhibitor apocynin (APOC) to improve NO-mediated responses in penile tissues of diabetic men and to block the deleterious effect on this response driven by the pharmacological stimulation (+) of PKC in non-diabetic tissues with 12,13-phorbol dibutyrate (PDBu).